Synthesis and Biological Evaluation of β-Phenylalanine Derivatives Containing Sulphonamide and Azole Moieties as Antiproliferative Candidates in Lung Cancer Models

Abstract

In this study, a series of novel β-phenylalanine derivatives were synthesised and evaluated for their anticancer activity. The 3-(4-methylbenzene-1-sulfonamido)-3-phenylpropanoic acid (2) was prepared using β-phenylalanine as a core scaffold. The β-amino acid derivative 2 was converted to the corresponding hydrazide 4, which enabled the development of structurally diverse heterocyclic derivatives including pyrrole 5, pyrazole 6, thiadiazole 8, oxadiazole 11, triazoles 9 and 12 with Schiff base analogues 13 and series1,2,4-triazolo [3,4-b][1,3,4]thiadiazines 14. These modifications were designed to enhance chemical stability, solubility, and biological activity. All compounds were initially screened for cytotoxicity against the A549 human lung adenocarcinoma cell line, identifying N-[3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxo-1-phenylpropyl]-4-methylbenzenesulfonamide (5) and (E)-N-{2-[4-[(4-chlorobenzylidene)amino]-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl]-1-phenylethyl}-4-methylbenzenesulfonamide (13b) as the most active. The two lead candidates were further evaluated in H69 and H69AR small cell lung cancer lines to assess activity in drug-sensitive and multidrug-resistant models. Schiff base 13b containing a 4-chlorophenyl moiety, retained potent antiproliferative activity in both H69 and H69AR cells, comparable to cisplatin, while compound 5 lost efficacy in the resistant phenotype. These findings suggest Schiff base derivative 13b may overcome drug resistance mechanisms, a limitation commonly encountered with standard chemotherapeutics such as doxorubicin. These results demonstrate the potential role of β-phenylalanine derivatives, azole-containing sulphonamides, as promising scaffolds for the development of novel anticancer agents, particularly in the context of lung cancer and drug-resistant tumours.

Keywords: 1,2,4-triazolo [3,4-b][1,3,4]thiadiazine; H69AR cells; anticancer agents; azole; oxadiazole; sulphanilamide; triazole; β-phenylalanine.

Scheme 1

Synthesis of compounds 2–9. (i) 4-TsCl, 15% Na₂CO_3(aq), Et₂O, r.t., 24 h, 20% HCl, pH~3; (ii) CH₃OH, H₂SO₄, reflux, 8 h; (iii) NH₂NH₂ ^. H₂O, 2-PrOH, reflux, 24 h; (iv) 2,4-pentanedione, 2-PrOH, AcOH, reflux, 2 h; (v) 2,5-hexanedione, 1,4-dioxane, AcOH, reflux 2 h; (vi) phenyl isocyanate or phenyl isothiocyanate, MeOH, reflux, 4 h; (vii) 80% H₂SO₄, 0 °C to r.t., 24 h, 5% NH₄OH, pH~7; (viii) 4% NaOH_(aq), reflux, 4 h, 20% HCl, pH~6.

Scheme 2

(13a) R = C₆H₅; (13b) R = 4-ClC₆H₄; (13c) R = 4-BrC₆H₄; (13d) R = 4-N(CH₃)₂C₆H₄; (13e) R = thiophen-2-yl; (13f) R = 3-indolyl; (13g) R = N-ethyl-9H-carbazol-3-yl; (14a) Ar = C₆H₅; (14b) Ar = 4-FC₆H₄; (14c) Ar = 4-ClC₆H₄; (14d) Ar = 4-BrC₆H₄. (i) CS₂, KOH, butan-1-ol, reflux 48 h; (ii) glacial AcOH, reflux, H₂O; (iii) NH₂NH₂ ^. H₂O; butan-1-ol, reflux, 72 h, glacial AcOH, reflux, H₂O; (iv) RCHO, 2-PrOH, reflux 8–24 h; (v) corresponding 2-bromoacetophenone, 1,4-dioxane, reflux, 8–18 h, 10% AcONa_(aq).

Figure 1

Non-proteogenic β-phenylalanine derivatives 2–14 demonstrated a structure-dependent antiproliferative activity in A549 cells. The cells were exposed to fixed 100 µM of compounds or doxorubicin (DOX), and cisplatin (CP) for 24 h, and the post-exposure viability was measured by MTT assay. The data is shown as mean ± SD of three experimental replicates.

Figure 2

The non-proteogenic β-phenylalanine derivatives 5 and 13b exhibited promising anticancer activity in both (A) drug-sensitive (H69) and (B) multidrug-resistant (H69AR) small cell lung cancer cell lines. Cells were treated with a fixed concentration (100 µM) of the compounds, doxorubicin (DOX), or cisplatin (CP) for 24 h, and cell viability was subsequently assessed using the MTT assay. Data are presented as the mean ± standard deviation (SD) of three independent experimental replicates. *** p < 0.002; **** p < 0.0001; ns—not significant.

Figure 3

The non-proteogenic β-phenylalanine derivatives 5 and 13b exhibited low cytotoxicity on non-cancerous HEK293 cells. Cells were treated with a fixed concentration (100 µM) of the compounds, doxorubicin (DOX), or cisplatin (CP) for 24 h, and cell viability was subsequently assessed using the MTT assay. Data are presented as the mean ± standard deviation (SD) of three independent experimental replicates. ** p < 0.01; *** p < 0.002; **** p < 0.0001; ns—not significant.

Figure 4

Non-proteogenic β-phenylalanine derivatives 5 and 13b induce cell death in a 3D A549 spheroid model. (A) Representative images of AO/PI-stained A549 spheroids following 24 h treatment with test compounds, illustrating live (green) and dead (red) cells. (B) Quantification of compound-induced cytotoxicity measured by lactate dehydrogenase (LDH) release into the culture medium. Data are presented as mean ± standard deviation (SD) from three independent experiments. *** p < 0.002; **** p < 0.0001.